The opium poppy P. somniferum is the plant from which opium is extracted. The opium poppy is the only commercially exploited poppy of the family Papaveraceae and is the principal source of natural opiates. The opium is extracted from latex harvested from the green seed pods. A further source of opiate alkaloids is the poppy straw which is the dried mature plant. P. somniferum is a source of clinically useful opiate alkaloids such as morphine, codeine, thebaine, noscapine [also known as narcotine] and papaverine. The clinical application of these opiate alkaloids and their derivates is broad having use as analgesics, cough suppressants and anti-spasmodics. Although not used as a pharmacological agent in its own right, thebaine is a particularly useful opiate which can be converted into a range of compounds such as hydrocodone, oxycodone, oxymorphone, nalbuphine naltrexone, buprenorphine and etorphine. These intermediates also have broad pharmaceutical applications. For example, oxycodone, oxymorphone and etorphine are widely used as an analgesic for moderate to severe pain and are often combined with other analgesics such as ibuprofen. Buprenorphine is used in the treatment of heroin addiction and chronic pain. Naltrexone is used in the treatment of alcohol and opiate addiction.
The use of thebaine in the production of these compounds is limited since thebaine is a minor component of the opiates extracted from poppy straw only accounting for approximately 0.5-2% of the opium extracted from dry straw. Mutant varieties of P. somniferum have been developed that can obtain thebaine and oripavine content of at least 50% by weight of the alkaloid combination of morphine, codeine, thebaine and oripavine; see WO98/02033. Alternative means to enhance the production of thebaine include the spraying of poppy plants with growth regulatory chemicals which inhibit alkaloid biosynthetic pathways to enhance the production of thebaine and other opiate alkaloids.
This disclosure relates to molecular analyses of gene expression in poppy cultivars that produce noscapine. Noscapine does not have significant analgesic properties but is used as a cough suppressant and is being investigated as an anti-cancer agent and in the treatment of stroke patients.
We have surprisingly found that certain varieties of poppy cultivars have genes that are unique to those cultivars that produce noscapine. We have cloned three genes that have homology to methyltransferases.
The first methyltransferase, PSMT1, exhibits sequence similarity to S-Adenosyl-L-Methionine:Scoulerine-9-O-Methyltransferase from Coptis japonica (Accession: Q39522.1, 61% identical) and from Thalictrum flavum (Accession: AAU20770.1, 59% identical). The protein from Coptis japonica has been characterised and shown to catalyse the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine to form tetrahydrocolumbamine (Sato et al. (1993) Phytochem. 32:659-664) which, in turn, serves as the precursor for the synthesis of most protoberberine alkaloids.
The second methyltransferase, PSMT2, exhibits sequence similarity to S-Adenosyl-L-Methionine:Norcoclaurine-6-O-Methyltransferase from Coptis japonica (Accession: Q9LEL6, 42% identical) which has been shown to catalyse the transfer of the S-methyl group of S-adenosyl-L-methionine to the 6-hydroxyl group of (S)-Norcoclaurine to form (S)-Coclaurine (Morishige et al. (2000) J. Biol. Chem. 275(30): 23398-23405).
The third methyltransferase, PSMT3, exhibits sequence similarity to S-Adenosyl-L-Methionine:Norcoclaurine-6-O-Methyltransferase from Papaver somniferum (Accession: AAQ01669, 80% identical) and Papaver bracteatum (Accession: ACO90232, 80% identical). The protein from Papaver somniferum has been characterised and shown to catalyse the transfer of the S-methyl group of S-adenosyl-L-methionine to to catalyse the transfer of the S-methyl group of S-adenosyl-L-methionine to the 6-hydroxyl group of (S)-Norcoclaurine to form (S)-Coclaurine (Ounaroon et al. (2003) 36:808-819).